In vitro gas production technique was used to evaluate ruminal fermentation characteristics of a mid-forage total mixed ration (TMR; containing 39.4% corn silage, 6.2% wheat straw and 54.4% concentrate) which was pre-treated with a commercial enzyme mixture (Natuzyme®) applied at the rate of 0.0, 0.84, 1.68 and 2.52 g/kg DM (E0.0, E0.84, E1.68 and E2.52, respectively) and at different times (0, 12 and 24 hr before to the start of the in vitro incubation, namely hr0, hr12 and hr24, respectively). Increasing levels of enzyme from 0.84 to 2.52 g/kg substrate DM linearly (P<0.001) increased gas production volume (GP96). In vitro dry matter disappearance (IVDMD) increased quadratically (P<0.05) with increasing level of enzyme and was highest at E1.68. Fermentation efficiency (FE) increased quadratically (P<0.001) with increasing level of enzyme and the highest (P<0.05) FE was at E0.84. Methane production at (t1/2) increased (P<0.05) linearly with increasing level of enzyme. Gas production volume (GP24) responded quadratically (P<0.001) as the time of enzyme administration increased from hr 0 to hr12 and hr24 and was highest at hr12. IVDMD and FE decreased linearly (P<0.001) as time of pre-incubation increased from 0h to 24 h. Results suggest that the enzyme used and the times of pretreatment are advantageous to improve in vitro fermentation of a mid-forage ration.

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